a, The experimental scheme for . MC tumor-bearing Rag1-/- mice (no B and T cells) were treated with oxaliplatin (weekly). One day after 1st oxaliplatin treatment, 5 × 106 T cells (negative selection) from WT mice immunized with MC cell lysate were adoptively transferred into tumor-bearing mice (4-5 mice/group), alone or in combination with 5 × 106 B cells from WT or Tgfbr2ΔB mice (purity 98%). After 2 more oxaliplatin cycles (3 weeks total), the mice were sacrificed and analyzed. b, Serum IgG analysis of above mice. c, Flow cytometric analysis of splenocytes after staining with CD45 and CD19 antibodies. All results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance. Statistical significance is given by *P, 0.05; **P, 0.01; ***P, 0.001. d-p, WT, Jh-/-, Iga-/- and Tgfbr2ΔB mice in the FVB background and WT, Pdl1/2-/-, Il10-/- and Iga-/- in the C57BL/6 background were analyzed for the distribution of immune markers. d, Spleen weights of WT, Jh-/- and Tgfbr2ΔB mice in the FVB background. e, Flow cytometry of splenocytes for the following markers: CD3 (left), CD8 (middle), CD4 (right), gated on the splenic CD45+ population. f, Absolute cell numbers of splenic CD3+ (left), CD8+ (middle), and CD4+ (right) cells are shown (percentage × cell count of whole spleen). g,h Flow cytometry for TNF and IFNγ in CD8+ cells from tumor-free WT, Jh-/-, Tgfbr2ΔB and Iga-/- mice (n=6-8) that were re-stimulated in vitro with PMA/ionomycin and the representative flow cytometry panels (e). i,j, Flow cytometry of splenocytes from WT and Tgfbr2ΔB for: CD19+IgM+ cells (i) and IgA (j) gated on the splenic CD45+ population. k-n, Flow cytometry of splenocytes from WT, Pdl1/2-/- and Il10-/- mice for: CD45+CD19+IgM+ cells (k), CD45+IgA+ cells (l), PD-L1 expression by CD19+IgM+ cells (m), and IL-10 expression by CD19+ cells (n). o,p, Serum IgA and IgG concentrations were analyzed in WT, Pdl1/2-/- and Il10-/- mice (n=4-5 mice/group). All results are means ± s.e.m. Mann-Whitney and t tests were used to calculate statistical significance shown as *P, 0.05; **P, 0.01; ***P, 0.001. The different gating strategies and staining controls are shown. q, Gating strategies for tumor-infiltrating lymphocytes: lymphocyte gate, dead cell exclusion, doublets exclusion, and gating on the CD45+ population. r, Flow cytometric analysis of IL-10 and IgA expression, gated on the CD45+ population: 1) isotype control (no staining), 2) non-stimulated splenocytes: showing IgA staining, but not IL-10. 3) stimulated splenocytes from Il10-/- mice showing IgA staining, but not IL-10. 4) stimulated splenocytes from WT mice showing IgA and IL-10 staining. s, Flow cytometric analysis of IL-10 and CD19 expression, gated on the CD19+B220+ population. left: stimulated cells from Il10-/- mice, showing B cell staining, but not IL-10; right: stimulated cells from WT mice showing B cell staining and IL-10 staining. t, Flow cytometric analysis of IL-10 and IgA expression, gated on the IgA+ population: left: stimulated cells from Il10-/- mice, showing IgA cell staining, but not IL-10; right: stimulated cells from WT mice showing IgA and IL-10 staining. These results confirm IL-10 production by IgA+ cells. u, Flow cytometric analysis of p-STAT1 staining with corresponding isotype control.